Treatments employing drug containing matrices for introduction into cellular lesion areas

ABSTRACT

Treatment of cellular disorders involving abnormal solid cellular growths involves introduction of cytotoxic reagents dispersed in a physiologically acceptable proteinaceous matrix into the solid cellular growth or area of an existing or removed solid cellular growth. Enhanced effectiveness of the drug is observed, with reduced cytotoxic effects on cells distant from the site of introduction. Other drugs may be included to enhance therapeutic gain and reduce adverse affects to normal tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part application of abandoned application Ser.No. 615,008, filed May 29, 1984, which disclosure is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The treatment of many cellular disorders, for example, tumors, involvesthe use of cytotoxic drugs. These drugs exert their activity in avariety of ways, usually interfering with a cellular function essentialfor the replication and/or viability of the cell. In many, if not most,instances, the drug is not specific for the unnatural cell, but rathertends to exert its effectiveness due to the more rapid proliferation ofthe abnormal cell, as compared to normal cells. While many organs of thebody of a mammalian host regenerate cells rather slowly, there are alsoother organs, particularly bone marrow, which involves rapidproliferation of stem cells. Therefore, the cytotoxic agents not onlycan detrimentally affect the slowly regenerating cells, but also have aparticularly pernicious effect on the immune system.

Despite the many disadvantages and side effects of employing thestrongly cytotoxic drugs, they have found extensive application, becausethey have provided positive results. However, there is substantialinterest in being able to employ the drugs in a manner which directstheir activity toward the abnormal cells, in an effort to protectsensitive normal cells, both in the vicinity of and distant from theabnormal cell growth, from the harmful effects of the drug.

2. Description of the Prior Art

U.S. Pat. Nos. 4,322,398; 4,347,234, 4,349,530; and 4,391,797 describeimplants and controlled release of drugs. Implantation of drugs inlesions is described in Maugh, Science (1981) 212:1128-1129; Macek etal., Abstracts of Immunology, 4109, p. 1053, Miyata et al., CancerResearch (1983) 43:4670-4675; McLaughlin et al., Cancer Research (1978)38:1311-1316; and Bier et al., Cancer (1979) 44:1194-1200.

SUMMARY OF THE INVENTION

Abnormal solid cellular growth, particularly tumors, or adjacent tissuethat may contain tumor cells, are treated by injecting into the abnormalgrowth area or tissue suspected of containing tumor cells a sufficientmount of a cytotoxic drug dispersed in a stable flowable proteinaceousmatrix. The resulting matrix substantially inhibits the migration of thedrug from the site of injection, so as to maintain the primary effect ofthe drug in the region of injection. Migration can be further inhibitedby the use of physiologically acceptable materials which enhance thebinding of the drug to the matrix or which modify cellular properties orphysiological responses to further regionalize the placement of drug atthe injection site.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel methods and compositions are provided for the chemotherapeutictreatment of solid abnormal tumors, cellular growth, or particularly,adjacent tissues which may contain abnormal tumor cells. The methodemploys a substantially uniform dispersion of a chemotherapeutic drug ina concentrated dispersion of a physiologically acceptable matrix,particularly a protein such as collagen, fibrinogen, or a derivativethereof, or other high molecular weight physiologically acceptablebiodegradable composition, dispersed in a minor amount of aphysiologically acceptable aqueous medium. The resulting amorphous massis injected into the lesion, e.g., tumor, or lesion area, e.g., adjacenttissue, or in those situations where the minor has been removed, tissueadjacent to the previously removed tumor. The proteinaceous matrix isflowable for injection, but provides for stable placement, once injectedinto the tissue. That is, once injected the proteinaceous matrix adheresto the tissue and does not migrate significantly. The treatment may beemployed with various solid tumors, including carcinomas and sarcomas.After injection, the drug is released into the immediate environment, soas to prevent substantial transportation of the drug to other sites,where its cytotoxic effect is undesirable. Thus, the circulating bloodlevel of the drug remains low. In this way an enhanced therapeutic gainis achieved, that is, the cytotoxic effect on malignant cells is greateras compared to susceptible normal cells.

Illustrative of the various diseased states or therapeutic modes inwhich the subject invention may find application are: (1) Neoplasms inwhich local recurrence is typical and drug bioavailability iscompromised, e.g., brain; (2) tumors in which suspected neoplastic cellsremain in the tumor bed following surgical resection, e.g., breast; (3)tumors which are poor candidates for surgical or radiation management,e.g., head, neck, prostate, etc.; (4) adjunctive tumor therapy incombination with physical or non-chemical treatments, e.g., radiationand/or hyperthermia; (5) hyperproliferative diseases refractory toconventional therapy, e.g., psoriasis; (6) concurrent with systemicchemotherapy; (7) concurrent with systemic rescue, e.g., methotrexate,plus collagen matrix intra-tumorally, leucovorin i.v.

The subject compositions are amorphous, injectable and viscous, so as tosubstantially retain a localized position without significant flow fromthe site of administration. The compositions can flow under moderatepressure, but will not move significantly after being positioned at aparticular site. The protein will be capable of binding the agentscovalently or non-covalently, without preventing their therapeuticeffect, while retaining the active agents at the site of introduction orretarding transfer of the active agents present from the site ofintroduction.

Preferably, the composition will be comprised of a significant amount ofthe matrix to provide the desired composition characteristics. Thematrix may be comprised of individual or in combination peptides orproteins, e.g., structural proteins such as collagen and fibrinogen, oralbumin or other protein which provides for stable placement, orcombinations thereof. Of particular interest is collagen, fibrinogen orderivative thereof.

Proteinaceous compositions having at least about 5 weight percent,preferably at least about 10 weight percent, and up to 50 weight percentor more, are of particular interest when used in combination withthrombin or its enzymatic equivalent. In this way fibrinogen isenzymatically modified to fibrin to enhance the non-migratory propertyof the composition while forming a matrix of fibrils to furtherstabilize the composition.

The thrombin may be mixed with fibrinogen containing proteinaceouscomposition from a time immediately prior to use or shortly afterinjection. The amount of thrombin of about 1 to 1000 IU/mg employed willgenerally range from about 0.1 to 10 weight percent of the fibrinogenpresent, depending upon the time of use, the rate desired for solidmatrix formation, the amount of other components, the effect of the drugon thrombin activity, and the like.

In addition to the matrix material will be one or more chemotherapeuticdrugs, and a physiologically acceptable aqueous medium in which theproteinaceous composition is dispersed and the drug may be dissolved,dispersed, or complexed with the collagen. Other materials arepreferably present to enhance the beneficial properties of the subjectcomposition.

The proteinaceous, particularly collagenous or fibrinogen-containing,material which is used may be derived from any mammalian host source,such as bovine, porcine or human, or may be prepared, as available, byother techniques, e.g. recombinant DNA techniques. The collagen employedmay be natural collagen or may be modified, such as tropocollagen,atropocollagen, or the like. The collagen may be non-immunogenic,immunogenic, or only slightly immunogenic.

Various methods of preparing collagen or derivatives thereof in purifiedform for administration to a mammalian host are known in the literature.These methods may be found in such patents as U.S. Pat. No. 3,949,073and references cited therein. Of interest is bovine collagen which ispurified and is obtained from young cows or calves. Purification willnormally involve dispersion or precipitation from various media, e.g.,dilute acetic acid. In some situations xenogeneic collagen is employedto enhance an immunogenic response in the area of injection orimmunogenic adjuvants may be employed.

A wide variety of chemotherapeutic drugs may be employed individually orin combination. The drugs may be bound or unbound to the matrix, throughsuch binding as complexation, salt formation, coordination complexes, orthe like, but any binding should not result in significant diminution ofthe physiological activity of the drug. Various drugs may be employedwhich are used in chemotherapy and act as alkylating agents, enzymeinhibitors, proliferation inhibitors, lytic agents, DNA synthesisinhibitors, membrane permeability modifiers, DNA intercalators,antimetabolites, or the like. Illustrative drugs include chlorambucil,melphalan, busulfan, carmustine, lomustine, streptozotocin, thiotepa,decarbazine methotrexate, 5-fluorouracil, cytarabine, azaribinemercaptopurine, thioguanine, vinblastine, vincristine, antinomycin D,adriamycin, bleomycin, mithramycin, mitomycin C, L-asparaginase,cisplatin, procarbazine, prednisone, prednisilone, triamicinolone,testosterone, estrogen, insulins, and hydroxyurea. See Carter andLivingston, Drugs Available to Treat Cancer. In Principles of CancerTreatment (Carter et al., eds.) Chapter 10, pp. 111-145, 1982,McGraw-Hill, Inc., N.Y. The drugs should not formnon-enzymatically-labile bonds with the matrix material resulting in theloss of their therapeutic effect.

The drugs may be used individually or in combination, depending upon thenature of the drug, the tumor, and whether cooperative action ispharmacologically indicated. The drug composition can be furthermodified, by modifying the drug, particularly by bonds which allow forenzymatic cleavage, e.g., hydrolysis, or by introducing materials intothe composition which will aid in the maintenance of the retention ofthe drug at the site of introduction.

Various techniques can be used for diminishing drug migration, forexample, by coupling the drug with specific ligands, such as lipids,phospholipids, peptides, amino acids, sugars, or the like. Thesemodifications will depend upon the individual drug, varying thesolubility of the drug in the aqueous medium and providing for covalentor non-covalent interactions with the protein. In addition, variousphysiologically acceptable bulking agents or concentrating agents may beemployed, which serve to provide for drug and protein interactions, withresulting reduction in the rate of drug release. Illustrative materialsinclude inorganic substances, such as hydroxyapatite and organicsubstances such as carbohydrates, e.g, agarose and cellulose.

Other drugs for use in combination with the chemotherapeutic agents aredrugs which retard the diffusion away of the chemotherapeutic agent, soas to reduce physiological insult and enhance therapeutic gain. Ofparticular interest are agents which restrict regional vasculature,either as to growth and/or passage opening, e.g., vasoconstrictive orsympathomimetic agents. These agents may include catechol amines, e.g.,epinephrine and nor-epinephrine ergot alkaloids, prostaglandins,angiotensin or the like. Other agents for affecting tissue architectureinclude enzymes which can injure the stroma, such as the peptidasespapain, chymopapain, trypsin, amylase, collagenase and chymotrypsin. Or,agents affecting cellular permeability may be employed, such asnon-ionic detergents, e.g., Tween 80, amphotericin B, dimethylsulfoxideand anaesthetics, such as procaine.

In addition, the drug(s) can be employed encapsulated in liposomes orother controlled rate release compositions, which are included in theproteinaceous composition, so as to provide for separate and distinctrates of release of the drug. In this way, multiphasic compositions canbe prepared, so as to provide for sustained release of the drug overlong periods of time. Formation of liposomes with inclusion of variousmaterials is described in Papahadjopoulos (1978) Annals of the N.Y.Academy of Science, 308; Gregoriadis and Allison (1980) Liposomes inBiological Systems, John Wiley and Sons, Leserman et al., Nature (1981)293:226-228; Barhet et al, Supramol. Struct. Cell Bio. Chem. (1981)16:243-258; and Heath et al., Science (1980) 255:8015-8018.Alternatively, other methods of encapsulation can be employed where thedrug is encapsulated in a biodegradable substance, where the rate ofrelease is related to the thickness of the biodegradable coat.

Besides using xenogeneic collagen, other materials may be included toenhance an immunogenic response,

e.g., proliferation and invasion of macrophage, helper T-cells, etc.Illustrative adjuvants include Corynebacterium parvum, BacillusCalmette-Guerin cell wall or cell wall skeleton preparations,Mycobacterium bovis strain, etc. See Miyata et al., Cancer Res. (1983)43:4670-4675; Bier et al., Arch. Otorhinolaryngol, (1982) 236:245-255;and Mehanjhlin et al., Cancer Res. (1978) 38:1311-1316, whose relevantdisclosure is incorporated herein by reference.

For enhancing cytotoxic activity various adjuvant materials may beincorporated into the matrix, such as radioactive pellet, e.g.,radionuclides Technicium or Iridium; radiation sensitizers, e.g.,misonidazole; repair inhibitors, e.g., methylated xanthines;bioreductive agents, which are activated only in hypoxic cells;immunomodifiers, such as interferons, lymphokines, such asinterleukin-2; tumor growth inhibitors, such as tumor necrosis factor,tumor growth factor-β, etc., and/or angiographic contrast media.

As already indicated, the ratio of dry materials in the composition mayvary widely. However, the amount of protein matrix material will usuallybe not less than 30% and not greater than about 95%, generally rangingfrom about from 40% to 90%, more usually ranging from about 50% to 90%by weight. Of this, preferably 10 to 100% will be collagen and/orfibrinogen. The chemotherapeutic drug(s) will normally be a liquid orsolid, or provided in solid form and a generally range from at leastabout 0.1% by weight to up to about 50% by weight, more usually beingfrom about 1% to 50% by weight, generally being from about 1% to 45% byweight of the proteinaceous material.

Other ancillary additives or agents will vary in total amount from about0.005 to 15, usually from about 0.01 to 10 weight percent of the dryweight of the total composition.

The composition is uniformly dispersed in a physiologically acceptableaqueous medium, such as saline, phosphate buffered saline, distilledwater, etc. The aqueous medium will be sufficient to provide for anamorphous dispersion capable of flowing under mild pressure. Usually,the liquid aqueous medium will be at least 90 weight percent of theentire composition, more usually at least 95% weight percent, and notmore than about 99.8 weight percent, usually not more than about 99.5weight percent, so as to provide a flowable mixture. The amount willvary depending upon the nature of the drug(s), the nature of the matrixmaterial, the presence of other materials, and the like. Theconcentration of protein in the aqueous medium will range from about 5to 75 mg/ml.

In addition to the major components, a number of minor components mayalso be included for a variety of purposes. These agents will for themost part impart properties which protect the stability of thecomposition, control the pH, or the like. Illustrative agents includephosphate or acetate buffers, methyl or propyl paraben, polyethyleneglycols, etc. These agents generally will be present in less than about2 weight percent of the total composition, usually less than about 1weight percent, and individually may vary from about 0.001 weightpercent to about 1 weight percent.

As already indicated, in some instances the drug will be encapsulatedparticularly in liposomes. Liposomes are prepared from a variety oflamellar-forming lipids including phospholipids, e.g.,phosphatidylcholine, phosphatidylethanolamine, etc., gangliosides,sphingomyelins, steroids, e.g., cholesterol, etc. Usually, the weight ofthe lipids in relation to the weight of drug will range from 1 to 5 L ofentrapped drug per mole of amphiphatic lipid.

The composition can be prepared by combining the various components in asterile environment. The matrix will be provided in a convenient form,usually admixed with at least a portion of the total aqueous medium tobe employed. The composition will be sufficiently workable that uponadmixture of the other agents a uniform dispersion can be obtained. Whencollagen or derivative thereof is used, the collagenous material willnormally be provided as a uniform dispersion of collagen fibrils in anaqueous medium, where the collagenous material will be from about 5mg/ml to not more than 100, usually not more than 75 mg/ml. The drug maythen be added to the collagenous dispersion with agitation to ensure theuniform dispersion of the drug in the resulting mixture. Othermaterials, as appropriate, may be added concomitantly or sequentially.After ensuring the uniform dispersion of the various components in themixture, the mixture may be sterilized and sealed in appropriatecontainer.

Sterilization will usually be achieved using aspetic conditions.

The subject composition can be used in the treatment of a wide varietyof neoplastic lesions. Illustrative tumors include carcinomas, sarcomasand melanomas, such as basal cell carcinoma, squamous cell carcinoma,melanoma, soft tissue sarcoma, solar keratoses, Kaposi's sarcoma,cutaneons malignant lymphoma, Bowen's disease, Wilm's tumor, hepatomas,colorectals cancer, brain tumors; mycosis fungoides, Hodgkins lymphoma,polycythemia Vera, chronic granulocytic leukemia, lymphomas, oat cellsarcoma, etc.

The subject composition will be administered to a tumor to provide acytotoxic amount of drug at the tumor site. The amount of cytotoxic drugadministered to the tumor site will generally range from about 0.1 to500, more usually about 0.5 to 300 mg/kg of host, depending upon thenature of the drug, size of tumor, and other considerations. Thevasoconstrictive agents will generally be present in from 1 to 50 weightpercent of the therapeutic agent. In view of the wide diversity oftumors, nature of tumors, effective concentrations of drug, relativemobility and the like, a definitive range cannot be specified. With eachdrug in each tumor, experience will provide an optimum level. One ormore administrations may be employed, depending upon the lifetime of thedrug at the tumor site and the response of the tumor to the drug.Administration may be by syringe, catheter or other convenient meansallowing for introduction of a flowable composition into the tumor.Administration may be every three days, weekly, or less frequent, suchas biweekly or at monthly intervals.

Illustrative of the manner of administration according to this inventionwould be administration of cis-diamino dichloro platinum. Drugconcentrations in the matrix may vary from 0.01 to 50 mg/ml. Injectionmay be at one or more sites depending on the size of the lesion. Needlesof about 1-2 mm diameter are convenient. For multiple injectiontemplates with predrilled holes may be employed. The drug dose willnormally be less than 100 mg/m² of patient.

The subject method finds particular advantage with tumors or lesionswhich are clinically relevant. The compositions provide therapeutic gainwith tumors greater than 100 mm³, more particularly, greater than 150mm³, in volume.

The subject method is also found to reduce local inflammation as aresult of the drug administration. Therefore, local or adjacent tissuesis less likely to be affected by the drug. Furthermore, due to the lowmigratory level of the drug from the site of placement, higher drugdosages can be administered to the site without adverse affects tonormal tissue distant from the placement site or to lymphocytes.

The subject method finds advantage in conjunction with other forms oftherapy. The lesions may be irradiated prior and/or subsequent to matrixadministration. Dose rates may vary from about 20 to 250 rad/min,usually 50 to 150 rad/min, depending on the lesion, period of exposure,and the like. Hyperthermia (heat) may be used as an adjunctivetreatment. Treatment will usually involve heating up to about andincluding 43° for about 5 to 100 min.

In order to demonstrate the subject invention, the followinginvestigations were performed. A transplantable experimental murinefibrosarcoma (2×10⁵ RIF-1 cells) was grown intradermally in the flank of5 month old female C3H mice (Bantin and Kingman, Fremont, CA).Cis-diamine dichloroplatinm (II) (cis-Pt) (Sigma Chemical Co., St.Louis, MO.) was dissolved in sterile saline at concentrations of 0.8,1.6 and 3.2 mg/ml and mixed 1:1 with bovine collagen (BC) (36 mg/ml) inPBS 20 mM phosphate, 140 mM NaCl (Collagen Corp., Palo Alto, CA). Dosesof 2, 4 and 8 mg/kg host of cis-Pt were delivered in 0.1 ml of thecollagenous drug mixture to the center of the tumor growing in the flank(intratumorally, i.t.), and the tumor measured. The growth of a seconduninjected tumor on the opposing flank of the same mouse was alsomeasured. In addition, cis-Pt dissolved in PBS without collagen wasadministered intraperitoneally (i.p.) to other tumor-bearing mice (4tumors/group) to monitor the effects on tumor growth of the drug withoutcollagen. Furthermore, the effect of bovine collagen on tumor growth wasalso studied by injection of 0.1 ml of collagen, (18 mg/ml) into theexperimental fibrosarcomas, as previously described. The growth of thetumors was monitored three times per week by caliper measurements ofthree perpendicular diameters of the tumor and calculating tumor volumefrom the formula

    V=π/6×D.sub.1 ×D.sub.2 ×D.sub.3.

The following Tables 1 and 2 indicate the results.

                  TABLE 1                                                         ______________________________________                                        Effect of cis-Pt-BC Fibrosarcoma Regrowth Delay                               Treatment                                                                             Route of   cis-Pt Dose                                                                             # Tumors                                                                             Regrowth                                  Group   Administration                                                                           (mg/kg)   Measured                                                                             Delay* (Days)                             ______________________________________                                        Untreated                                                                             i.p.       --        4       6.3 ± 0.3**                           (PBS)                                                                         cis-Pt  i.p.       2         4      7.2 ± 0.1                              cis-Pt-BC                                                                             i.t.       2         4      9.0 ± 1.1                              cis-Pt  i.p.       4         6      9.1 ± 0.7                              cis-Pt-BC                                                                             i.t.       4         4      9.9 ± 0.3                              cis-Pt  i.p.       8         6      9.9 ± 1.0                              cis-Pt-BC                                                                             i.t.       8         4      12.7 ± 1.2                             ______________________________________                                         *Regrowth Delay determined as the time (days) for tumors to grow to four      times their initial treatment volume (150 mm.sup.3). Increasing values        indicate enchanced therapeutic effect.                                        **Mean ± S.E.                                                         

                  TABLE 2                                                         ______________________________________                                        Effect of cis-Pt-BC on the Growth of Uninjected                               Contralateral Fibrosarcoma                                                    Treatment                                                                             Route of   cis-Pt Dose                                                                             # Tumors                                                                             Regrowth                                  Group   Administration                                                                           (mg/kg)   Measured                                                                             Delay* (Days)                             ______________________________________                                        Untreated                                                                             i.p.       --        4       6.3 ± 0.3**                           (PBS)                                                                         cis-Pt  i.p.       2         4      7.2 ± 0.1                              cis-Pt  i.t.       2         4      9.5 ± 0.5                              uninjected                                                                            --         --        4      9.3 ± 1.2                              contra                                                                        cis-Pt-BC                                                                             i.t.       2         4      9.0 ± 1.1                              uninjected                                                                            --         --        4      7.2 ± 0.4                              contra                                                                        cis-Pt  i.p.       4         6      9.1 ± 0.7                              cis-Pt  i.t.       4         4      10.3 ± 0.7                             uninjected                                                                            --         --        4      8.9 ± 0.7                              contra                                                                        cis-Pt-BC                                                                             i.t.       4         4      9.9 ± 0.3                              uninjected                                                                            --         --        4      7.4 ± 0.6                              contra                                                                        cis-Pt  i.p.       8         6      9.9 ± 1.0                              cis-Pt  i.t.       8         4      11.5 ± 0.04                            uninjected                                                                            --         --        4      9.9 ± 0.9                              contra                                                                        cis-Pt-BC                                                                             i.t.       8         4      12.7 ± 1.3                             uninjected                                                                            --         --        4      9.0 ± 1.1                              contra                                                                        ______________________________________                                         *Regrowth Delay determined as the time (days) for tumors to grow to four      times their initial treatment volume (150 mm.sup.3).                          **Mean ± S.E.                                                         

In the next study 5-fluorouracil (5-FU) (Sigma Chem. Co., St Louis, Mo.)with and without epinephrine (Sigma) suspended in saline by sonication(60 mg/ml) and mixed 1:1 with bovine collagen (BC) (Collagen Corp., PaloAlto, CA) (36 mg/ml) or normal saline. The subjects were 25 gm 12week-old female C3H/He mice (Bantin and Kingman, Fremont, CA) bearingthe transplatable experimental murine fibrosarcoma propagatedintradermally as previously described.

When the tumors reached a volume of 150 mm³, the mice were assignedrandomly to the following groups (4-6 mice per group): (1) untreatedcontrols; (2) 5-FU (100 mg/kg), i.p., 0.1 ml/mouse; (3) 5-FU (100mg/kg), i.t., 0.1 ml/tumor; (4) 5-FU-BC (100 m.g 5-FU/kg dispersed in BC(18 mg/ml)), i.t., 0.1 ml/tumor, 5-FU-EPI-BC (100 mg 5-FU/kg) 5 mgEPI/kg dispersed in BC (18 mg/ml), i.t., 0.1 ml/tumor.

On day four post-treatment, white blood cells (wbc) were counted bysampling blood from the tail, dilution in Turk's solution and countingin a hemocytometer. On day eight post-treatment, skin reaction isoverlying tissue was graded for untoward response.

The following Table 3 indicates the results.

                  TABLE 3                                                         ______________________________________                                        Effect of 5-Fluorouracil (5-FU) (100 mg/kg) - Bovine                          Collagen (BC) ± Epinephrine (EPI) (5 mg/kg) on Tumor                       Growth and Normal Tissue Response**                                                               Untreated                                                 Experimental                                                                          Tumor       Contralateral                                                                           White Blood                                     Group (4-6                                                                            Regrowth    Regrowth  Cells/mm.sup.3                                                                        Skin                                    mice/group)                                                                           Delay (days)                                                                              Delay (days)                                                                            (×10.sup.3)                                                                     Reaction*                               ______________________________________                                        Untreated                                                                               6.3 ± 0.7***                                                                          6.3 ± 0.7                                                                           7.9 ± 1.6                                                                          1.0 ± 0.4                            Controls                                                                      5-FU i.p.                                                                             13.1 ± 1.4                                                                             13.1 ± 1.4                                                                           4.5 ± 0.6                                                                          0.7 ± 0.4                            5-FU i.t.                                                                             14.5 ± 0.9                                                                             11.1 ± 0.6                                                                           3.5 ± 0.5                                                                          0                                       5-FU-BC i.t.                                                                          15.1 ± 3.3                                                                              8.6 ± 0.5                                                                           5.4 ± 2.2                                                                          1.3 ± 0.5                            5-FU-EPI-                                                                             17.7 ± 1.7                                                                             12.5 ± 1.2                                                                           5.0 ± 1.5                                                                          0                                       BC i.t.                                                                       ______________________________________                                         *Skin reaction. Evaluation of the skin overlying the tumor on Day 8 post      injection.                                                                    The skin reaction is scored as follows:                                       0 = no effect;                                                                1 = superficial inflammation;                                                 2 = scab;                                                                     3 = ulcer.                                                                    **5FU-EPI i.t. was lethal to the mouse.                                       ***mean ± S.E.                                                        

In the next study doxorubicin-HCl (ADM) (Adriamycin) was studied usingthe above-described protocol. The adriamycin in distilled water (4.45mg/ml) was mixed with 36 mg/ml bovine collagen (BC) 1:1 to yield acomposition ratio of 2.25 mg adriamycin:18 mg BC/ml. Intraperitonealinjection of 15 mg/kg of adriamycin was lethal to 75% of the mice, whileintratumoral injection was found to be non-toxic.

The following Table 4 indicates the results.

                                      TABLE                                       __________________________________________________________________________    Effect of Adriamycin (ADM) (15 mg/kg) - Bovine Collagen (BC)                  on Acute Animal Toxicity, Tumor Growth and Normal Tissue Response                      Animal                                                                             Tumor Regrowth Delay.sup.2                                                               White Blood                                          Experimental Group                                                                     Survival.sup.1                                                                     Treated/Contralateral.sup.3                                                              Cells/mm.sup.3 4                                                                    Skin                                           (4-6 mice/group)                                                                       (%)  (days)     (×10.sup.3)                                                                   Reaction.sup.5                                 __________________________________________________________________________    Untreated Controls                                                                     100  5.4 ± 0.2/5.4 ± 0.2                                                                10.2 ± 1.7                                                                       1.3 ± 0.4                                   Free ADM i.p.                                                                           25  10.6 ± 1.6/10.6 ± 1.6                                                              3.4   1.5 ± 0.5                                   Free ADM i.t.                                                                          100  13.7 ± 1.7/8.3 ± 0.75                                                              4.1 ± 0.69                                                                       2.0                                            ADM-BC i.t.                                                                            100  10.6 ± 0.9/7.8 ± 1.3                                                               6.8 ± 0.71                                                                       1.8 ± 0.2                                   __________________________________________________________________________     .sup.1 Animal survival after injection of ADM 15 mg/kg. Deaths usually        resulted within 2 days after injection.                                       .sup.2 Tumor Regrowth Delay (RD). Time (days) required for tumors to grow     to 3× its original treatment volume (˜150 mm.sup.3).              Increasing RD indicate increased tumor cell killing.                          .sup.3 Contralateral Tumor RD (CRD). Time (days) required for untreated       contralateral tumors to grow to 3× its original treatment volume        (˜150 mm.sup.3). Decreasing RD indicate enhanced regionalization of     drug injected when compared to RD.                                            .sup.4 White Blood Cells measured on Day 4 post injection by sampling fro     the tail of treatment mice.                                                   .sup.3 Skin reaction. Evaluation of the skin overlying the tumor on Day 8     post injection. The skin reaction is scored as follows:                       0 = no effect;                                                                1 = superficial inflammation;                                                 2 = scab;                                                                     3 = ulcer;                                                               

In the next study vincristine (VCR) was dissolved in saline (0.6 mg/ml)by sonication and mixed 1:1 with bovine collagen (36 mg/ml). Otherwise,the procedure was the same. The following Table 5 indicates the results.

                  TABLE 5                                                         ______________________________________                                        Effect of Vincristine (VCR) (2 mg/kg) - Bovine Collagen                       (BC) on Tumor Growth and Normal Tissue Response                                                  Untreated                                                  Experimental                                                                          Tumor      Contralateral                                                                           White Blood                                      Group (4-6                                                                            Regrowth   Regrowth  Cells/mm.sup.3                                                                        Skin                                     mice/group)                                                                           Delay (days)                                                                             Delay (days)                                                                            (×10.sup.3)                                                                     Reaction                                 ______________________________________                                        Untreated                                                                              5.3 ± 0.2                                                                            5.3 ± 0.2                                                                            10.2 ± 1.7                                                                         1.3 ± 0.4                             Controls                                                                      VCR i.p.                                                                              10.6 ± 2.0                                                                            10.6 ± 2.0                                                                           7.4 ± 1.3                                                                          0.6 ± 0.4                             VCR-BC i.t.                                                                           10.2 ± 1.3                                                                            7.6 ± 1.1                                                                            9.4 ± 2.9                                                                          1.2 ± 0.5                             ______________________________________                                    

In the next study a combination of bleomycin sulfate (Sigma ChemicalCo., St. Louis, MO) (15 mg/kg) and epinephrine (5 mg/kg) employed in abovine collagen composition were evaluated for antitumor effect in thetransplantable experimental murine fibrosarcoma model previouslydescribed. The following Table 6 provides the results.

                  TABLE 6                                                         ______________________________________                                        Effect of Bleomycin Sulfate (BLM) (15 mg/kg) - Bovine                         Collagen (BC) ± Epinephrine (EPI) (5 mg/kg) on Tumor                       Growth and Normal Tissue Response                                                                Untreated                                                  Experimental                                                                          Tumor      Contralateral                                                                           White Blood                                      Group (4-6                                                                            Regrowth   Regrowth  Cells/mm.sup.3                                                                        Skin                                     mice/group)                                                                           Delay (days)                                                                             Delay (days)                                                                            (×10.sup.3)                                                                     Reaction                                 ______________________________________                                        Untreated                                                                              6.3 ± 0.7*                                                                           6.3 ± 0.7                                                                            7.9 ± 1.6                                                                          1.0 ± 0.4                             Controls                                                                      BLM i.p.                                                                              7.5 ± 0.9                                                                             7.5 ± 0.9                                                                            10.8 ± 1.8                                                                         1.5 ± 0.3                             BLM i.t.                                                                              8.9 ± 0.6                                                                             7.0 ± 0.7                                                                            7.0 ± 1.3                                                                          2.3 ± 0.3                             BLM-BC i.t.                                                                           9.4 ± 0.9                                                                             7.2 ± 0.1                                                                            8.0 ± 1.5                                                                          1.8 ± 0.3                             BLM-EPI-                                                                              9.7 ± 0.6                                                                             7.2 ± 1.2                                                                            23.3 ± 10.7                                                                        1.5 ± 0.5                             BC i.t.                                                                       ______________________________________                                         *mean ± S.E.                                                          

In another experiment the curative potential of drug matrix formulationswas evaluated in the experimental murine fibrosarcoma model. Briefly,female C3H/He mice bearing a single experimental tumor produced aspreviously described were treated at weekly intervals with formulationscontaining 5-fluorouracil (50 mg/kg); bovine collagen BC (CollagenCorp., Palo Alto, CA); epinephrine (Sigma Chemical Co., St. Louis, MO);and PBS. WBC's were determined on Day 4 following each treatment cycleand skin reaction on Day 3 after each treatment cycle. Treatment wasdiscontinued for all grops when 3 of 4 experiment groups reach 4×initialtumor volume. When tumors reached a volume of 150 mm³ the mice wererandomly assigned to the following groups:

1. Untreated controls

2. 5-FU-PBS i.p.; 5-FU (23 mg/ml) was combined 1:1 with PBS; 0.1 mlinjected/mouse i.p.

3. 5-FU-PBS i.t.; 5-FU (23 mg/ml) was combined 1:1 with PBS; 0.1 mlinjected/tumor i.t.

4. 5-FU-BC i.t.; 5-FU (23 mg/ml) was combined 1:1 with bovine collagen(36 mg/ml); 0.1 ml injected i.t.

5. 5-FU-BC-epi i.t.; 5-FU (23 mg/ml) was combined 1:1 with a bovinecollagen (36 mg/ml) containing epinephrine (2.4 mg/ml); 0.1 ml injectedi.t.

The results are shown in below in Table 7.

                  TABLE 7                                                         ______________________________________                                        Effect of 5-Fluorouracil (5-FU 50 mg/kg Administered on                       Days 0, 8 and 16) - Bovine collagen ± Epinephrine (5 mg/kg)                on Tumor Growth and Normal Tissue                                                       Tumor     Contra-          Skin                                     Experimental                                                                            Regrowth  lateral RD                                                                              WBC    Reaction                                 Group     4× (days)                                                                         4× (days)                                                                         D-12   D-8                                      ______________________________________                                        Untreated  6.3 ± 1.1                                                                           --         79 ± 13                                                                          2.6 ± 0.1                             Controls                                                                      5-FU-PBS i.p.                                                                           10.3 ± 1.3                                                                           --        109 ± 28                                                                          1.2 ± 1.1                             5-FU-PBS i.t.                                                                           14.9 ± 3.8                                                                           --        107 ± 25                                                                          1.0 ± 0.6                             5-FU-BC i.t.                                                                            11.2 ± 4.2                                                                           --        120 ± 21                                                                          1.6 ± 0.5                             5-FU-BC-EPI i.t.                                                                        26.0 ± 1.7                                                                           --        62 ± 6                                                                            1.4 ± 0.5                             ______________________________________                                    

The results indicate that epinephrine (5 mg/Kg) used as a vascoactivemodifier with low dose 5-FM-CM drug-matrix administered intratumorally(i.t.) in three weekly injections enhanced the antitumor effect of 5-FUby a factor of 2-2.5 with respect to i.p. treated minor regrowth delay.

In another experiment the influence of matrix composition on antitumoractivity of 5-fluorouracil (100 mg/kg) was evaluated in the experimentalmurine fibrosarcoma model previously described. 5-FU (Sigma ChemicalCo., St. Louis, MO) was combined as described below with bovine collagenBC (Collagen Corp., Palo Alto, CA); bovine fibronogen (95% clottable,Sigma); bovine thrombin (2000NIH units/mg, Sigma) and Ringer's SolutionFor Injection (RFI, Abbott Labs., North Chicago, IL). When tumorsreached a volume of 150 mm³ the mice (Bantin and Kingman, Fremont, CA)were assigned randomly to the following groups, (3-4 mice/group):

1. Untreated controls

2. Fibrinogen 30 mg/ml; fibrinogen (60 mg/ml) dispersed 1:1 with RFIcontaining 10 μl thrombin (1 NIH unit of activity/ml); 0.1 ml i.t.

3. 5-FU-Fibrinogen: 5-FU (36 mg/ml) combined 1:1 with the fibrinogenpreparation described in 2 above, 0.1 ml i.t.

4. 5-FU-BC-Fibrinogen: 5-FU (36 mg/ml) combined 1:1 with a fibrinogen-BCpreparation consisting of fibrinogen (30 mg/ml); BC (36 mg/ml) dispersedin RFI containing 1 NIH unit of thrombin activity/ml, 0.1 ml i.t.

The results are summarized in the following Table 8.

                  TABLE 8                                                         ______________________________________                                        Effect of Matrix Composition on Activity of 5-Fluorouracil (100 mg/kg)                  Tumor                                                               Experimental                                                                            Regrowth  Untreated                                                 Group     Delay 4×                                                                          Contralateral                                                                            WBC/   Skin                                    3-4 mice/grp                                                                            (days)    RD 4× (days)                                                                       mm.sup.3 × 10.sup.3                                                            Reaction                                ______________________________________                                        Untreated 6.3 ± 1.1                                                                            6.3 ± 1.1                                                                              79 ± 13                                                                          2.6 ± 0.1                            Controls                                                                      5-FU-Fib (30 mg/                                                                        11.9 ± 1.7                                                                           7.7 ± 0.9                                                                             147 ± 38                                                                          1.3 ± 0.6                            ml)                                                                           Fib (30 mg/ml)                                                                          5.5 ± 0.8                                                                            5.9 ± 0.5                                                                             276 ± 64                                                                          2.0 ± 0.0                            5-FU-Fib (15 mg/                                                                        9.1 ± 0.8                                                                            8.2 ± 0.6                                                                             168 ± 27                                                                          2.0 ± 0.0                            ml) - BC (18 mg/                                                              ml)                                                                           ______________________________________                                    

As evidenced from the above results, substantial advantages are obtainedin therapeutic gain, both in the presence or absence of ancillaryagents, when the therapeutic drugs are formulated as a flowable matrixin collagen and implanted in the lesion. The formulation retains thehigh chemotherapeutic activity of the chemotherapeutic agent, whilesubstantially reducing the cytotoxic effect on white blood cells andinflammatory activity on adjacent epidermal tissue.

The evidence for reduced systemic exposure is apparent from the lack ofimmunosuppression, the relative absence of tumor regression on thecontralateral uninjected tumor, and by the relative lack of untowardeffect on overlaying normal tissue.

In the next experiment 5-fluorouracil-matrix implant in combination withX-rays was evaluated. Single RIF-1 tumors were grown on the back offemale C3H mice (12-16 weeks) (Bantin and Kingman, Fremont, CA) aspreviously described. When the tumors reached volumes of 150 mm³, theywere divided into the following groups, (4-6 mice/group):

1. Untreated controls

2. X-rays (1000 rad.) alone

3. Collagen-matrix (CM) i.t. 5 min. before X-rays

4. 5-Fluorouracil (5-FU) (75 mg/Kg) i.p.

5. 5-FU-CM (75 mg/KG) i.t.

6. 5-FU i.p. 5 min before X-rays

7. 5-FU-CM i.t. 5 min before X-rays

Tumor bearing mice were irradiated in lead jigs exposing only the tumorand overlying skin with a 250 kVp X-ray machine at a dose rate of 120rad/min. The tumors of the treated and untreated mice were measuredthree times per week and assayed for regrowth delay and skin reactionsas previously described.

The results are set forth in the following Table 9:

                  TABLE 9                                                         ______________________________________                                        Effect of 5-Fluorouracil (5-FU) (75 mg/Kg) - Collagen Matrix (CM)             (30 mg/ml) Intralesional (i.t.) Implants in Conjunction with X-rays           (1000 rad) on RIF-1 Tumor Regrowth Delay                                                      2× Tumor Regrowth                                                                    Skin                                             Experimental Group                                                                            Delay (days) Reaction                                         ______________________________________                                        Untreated Controls                                                                            3.1 ± 0.4 2                                                5-FU (i.p.)     8.5 ± 1.1 2                                                5-FU-CM (i.t.)  6.3 ± 0.5 2                                                X-rays alone    6.9 ± 0.3 2                                                CM (i.t.) + X-rays                                                                            6.0 ± 0.3 2                                                5-FU (i.p.) + X-rays                                                                          12.6 ± 2.8                                                                              2                                                5-FU-CM (i.t.) + X-rays                                                                       15.5 ± 0.9                                                                              2                                                ______________________________________                                    

The results indicate that in a combined modality setting intralesional(i.t.) administration of 5-FU-CM in conjunction with X-rays iscomparable to X-rays with systemic (i.p.) 5-FU in terms of regrowthdelay.

In the next study cis-DDP-matrix (DDP=cis-Pt) implant in combinationwith hyperthermia was evaluated. Single RIF-1 tumors were grown on theback of female C3H mice as previously described. When the tumors reachedvolumes of 150 mm³, they were divided into the following groups (4-6mice/group):

1. Untreated controls

2. Hyperthermia (43° C., 30 min) alone

3. Hyperthermia+collagen-matrix (CM)+epinephrine (epi) (2 mg/Kg)

4. cis-DDP (6 mg/Kg) i.p.

5. cis-DDP-CM-epi (i.t.)

6. cis-DDP (i.p.) 30 min before hyperthermia

7. cis-DDP-CM-epi (i.t.) 30 min before hyperthermia.

The tumor bearing mice were heated in a precision water bath with 30gauge thermistor thermometry (±0.2° C.). The tumors of the treated anduntreated mice were measured three times per week and assayed forregrowth delay as previously described.

The following Table 10 indicates the results:

                  TABLE 10                                                        ______________________________________                                        Effect of Local Hyperthermia (43° C. 30 min) on cis-DDP (6 mg/Kg)      Collagen Matrix (CM) (30 mg/ml) - Epinephrine (Epi) (2 mg/Kg)                 Intralesional (i.t.) Implants on RIF-1 Regrowth Delay                                             Tumor Regrowth Delay (2×)                           Experimental Group  (2×) (days)                                         ______________________________________                                        Untreated Controls  3.5 ± 0.1                                              Hyperthermia alone  7.9 ± 1.3                                              Hyperthermia + CM-Epi (i.t.)                                                                      8.5 ± 0.5                                              cis-DDP (i.p.)      6.5 ± 1.5                                              cis-DDP-CM-Epi (i.t.)                                                                             10.0 ± 0.1                                             cis-DDP (i.p.) 30 min before hyperthermia                                                         8.9 ± 0.8                                              cis-DDP-CM-Epi (i.t.) 30 min before                                                               21.5 ± 2.3                                             hyperthermia                                                                  ______________________________________                                    

The results indicate that local hyperthermia can enhance the effect ofcollagen matrix associated cis-DDP administered intratumorally. Collagenmatrix (CM) with epinephrine (i.t.) alone with hyperthermia did notincrease the antitumor effect of hyperthermia.

In accordance with the subject invention, improved neoplastic therapy isachieved by applying to an oncogenic lesion a composition comprising achemotherapeutic drug composition, by itself or in combination with avasoconstrictive agent uniformly dispersed in a collagenous aqueousdispersion and introducing the viscous amorphous mixture into thelesion. It is found that by employing the drug-collagenous composition,greatly enhanced localized drug concentration can be achieved. Inaddition, in view of the significant cytotoxic effects of drugs employedin chemotherapy, systemic exposure is substantially diminished.Therefore, high levels of cytotoxic drugs can be employed at the site ofinterest, while the remainder of the host is not exposed to significantlevels of the drug. In addition, the drug pharmacokinetics are modified,due to modifications of the drug and/or interactions with the collagen,providing for a low level of the drug in the circulating blood. Finally,the lifetime of the drug can be extended due to protection by thecollagenous material, reducing the rate of metabolic inactivation.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A proteinaceous composition comprising from 30%to 95% of collagen and/or fibrinogen dispersed in an aqueous medium asan amorphous flowable mass at a concentration of from about 5 to 75mg/ml and from about 0.1 to 50 weight percent based on said collagenand/or fibrinogen of a cytotoxic drug .Iadd.or proliferationinhibitor.Iaddend..Badd., wherein when said proteinaceous compositioncomprises collagen, said collagen is present as a uniform dispersion ofcollagen fibrils.Baddend..
 2. A composition according to claim 1,wherein said cytotoxic drug is cis-platinum, adriamycin, bleomycin,5-fluorouracil or vincristine.
 3. A .Badd.proteinaceous.Baddend.composition . .. .according to claim 1, having.!..!..Badd.comprising from 30% to 95% of collagen and/or fibrinogen dispersedin an aqueous medium as an amorphous flowable mass at a concentration offrom about 5 to 75 mg/ml; from about 0.1 to 50 weight percent based onsaid collagen and/or fibrinogen of a cytotoxic drug or proliferationinhibitor and .Baddend.a vasoconstrictive amount of a vasoconstrictivedrug.
 4. A composition according to claim 3, wherein said . ...vasoconstrictor.!..!. .Badd.vasoconstrictive .Baddend.drug isepinephrine or nor-epinephrine.
 5. A method for treating a neoplastic.Iadd.or hyperproliferative .Iaddend.lesion or surrounding tissue whichcomprises:introducing at the site of said lesion a proteinaceous matrixcomposition capable of stable placement, consisting essentially . .or.!..Iadd.of a .Iaddend.physiologically acceptable matrix forming collagen,fibrinogen or combination thereof, dispersed in an aqueous medium as anamorphous flowable mass including at least one cytotoxic drug .Iadd.orproliferation inhibitor .Iaddend.uniformly dispersed in saidcomposition. .;.!.. .. .:.!..!..Badd.;.Baddend. whereby said . ...drug.!..!. .Badd.cytotoxic drug or proliferation inhibitor .Baddend.isslowly released into the . .. .immediate environment.!..!..Badd.neoplastic or hyperproliferative lesion or surrounding tissue.Baddend.avoiding significant levels of . .. .the drug.!..!. .Badd.saidcytotoxic drug or proliferation inhibitor .Baddend.at sites distant fromthe site of introduction.
 6. A method according to claim 5, wherein saidproteinaceous composition is a collagen fibril dispersion.
 7. A methodaccording to claim 6, wherein said . .. .drug.!..!. .Badd.cytotoxic drugor proliferation inhibitor .Baddend.is at least one of cis-platinum,adriamycin, . .. .5fluorouracil.!..!. .Badd.5-fluorouracil.Baddend.,bleomycin, vincristine, or methotrexate.
 8. A method according to claim7, wherein said composition includes a sufficient amount of a . ...vasoconstrictor.!..!. .Badd.vasoconstrictive drug .Baddend.to constrictcapillaries in the vicinity of said lesion.
 9. A method according toclaim 8, wherein said . .. .vasoconstrictor.!..!. .Badd.vasoconstrictivedrug .Baddend.is epinephrine or nor-epinephrine.
 10. A method accordingto claim 7, wherein said . .. .drug.!..!. .Badd.cytotoxic drug orproliferation inhibitor .Baddend.is cis-platinum.
 11. A method accordingto claim 7, wherein said . .. .drug.!..!. .Badd.cytotoxic drug orproliferation inhibitor .Baddend.is 5-fluorouracil.
 12. A methodaccording to claim 5, comprising the additional step of treating saidlesion . .. .size.!..!. .Badd.site .Baddend.with radiation or heat..Iadd.13. A proteinaceous composition comprising from 30% to 95% ofcollagen and/or fibrinogen dispersed in an aqueous medium as anamorphous flowable mass at a concentration of from about 5 to 75 mg/mland from about 0.1 to 50 weight percent based on said collagen and/orfibrinogen of a cytotoxic drug.Iaddend..Badd., wherein when saidproteinaceous composition comprises collagen, said collagen is presentas a uniform dispersion of collagen fibrils. .Baddend..Iadd.14. Aproteinaceous composition comprising from 30% to 95% of collagen and/orfibrinogen dispersed in an aqueous medium as an amorphous flowable massat a concentration of from about 5 to 75 mg/ml and from about 0.1 to 50weight percent based on said collagen and/or fibrinogen of aproliferation inhibitor.Iaddend..Badd., wherein when said proteinaceouscomposition comprises collagen, said collagen is present as a uniformdispersion of collagen fibrils. .Baddend..Iadd.15. A method for treatinga neoplastic lesion or surrounding tissue which comprises:introducing atthe site of said lesion a proteinaceous matrix composition capable ofstable placement consisting essentially of a physiologically acceptablematrix forming collagen, fibrinogen or combination thereof, dispersed inan aqueous medium as an amorphous flowable mass, including at least onecytotoxic drug or proliferation inhibitor uniformly dispersed in saidcomposition; whereby said .Iadd.. .. .drug.!..!..Iaddend..Badd.cytotoxic drug or proliferation inhibitor .Baddend..Iadd.is slowlyreleased into the .Iadd.. .. .immediate environment.!..!..Iaddend..Badd.neoplastic lesion or surrounding tissue .Baddend..Iadd.avoidingsignificant levels of .Iaddend..Iadd.. .. .the drug.!..!..Iaddend..Badd.said cytotoxic drug or proliferation inhibitor .Baddend..Iadd.atsites distant from the site of introduction. .Iaddend..Iadd.16. A methodfor treating a hyperproliferative lesion or surrounding tissue whichcomprises:introducing at the site of said lesion a proteinaceous matrixcomposition capable of stable placement consisting essentially of aphysiologically acceptable matrix forming collagen, fibrinogen orcombination thereof, dispersed in an aqueous medium as an amorphousflowable mass, including at least one cytotoxic drug or proliferationinhibitor uniformly dispersed in said composition; whereby said.Iaddend..Iadd.. .. .drug.!..!..Iaddend. .Badd.cytotoxic drug orproliferation inhibitor .Baddend..Iadd.is slowly released into the.Iadd.. .. .immediate environment.!..!..Iaddend..Badd.hyperproliferative lesion or surrounding tissue.Baddend..Iadd.avoiding significant levels of .Iaddend..Iadd.. .. .thedrug.!..!..Iaddend. .Badd.said cytotoxic drug or proliferation inhibitor.Baddend..Iadd.at sites distant from the site of introduction. .Iaddend.